It has been hypothesized that light-at-night (LAN) in modern societies might be partially responsible for the elevated risk of breast cancer observed in industrialized nations, and shift work is regarded as the most extreme form of LAN exposure. In October 2007, the International Agency for Research on Cancer (IARC) assembled an expert panel to assess the role of shift work in human cancer development by reviewing the evidence from laboratory and epidemiological studies. This expert panel concluded that "shift work that involves circadian disruption is probably carcinogenic to humans" (Lancet Oncology, 2007). Our group has completed pioneering work on the molecular epidemiology of circadian genes, and we have demonstrated that both genetic variants and methylation changes in circadian genes predispose individuals to breast cancer. However, whether shift work may contribute to epigenetic changes is still an unanswered question. Considering that millions of women worldwide are being exposed to ill-timed light, particularly through occupations involving shift work, there exists an urgent need to systematically study the biological impact of shift work, including the epigenetic consequences of this exposure. Here, we propose a human study to examine both global and locus-specific methylation changes that may result from shift work. In the proposed study, a total of 600 female subjects will be selected from an established Danish prospective cohort (300 subjects with no history of shift work and 300 long-term (10+ years) shift workers). These participants have archived blood samples available, which will be used for DNA extraction and subsequent epigenetic analysis, in order to determine the impact of shift work on global DNA methylation, as well as methylation in the promoter regions of the core circadian genes. In light of the recent IARC conclusion, along with an increasing body of epidemiological and biological evidence, this exploratory study will extend our previous discoveries and break new ground toward a more comprehensive understanding of the clock-cancer connection. More importantly, because of the preventable nature of some epigenetic events, the results obtained from this study will have considerable public health implications and could lead to new strategies for breast cancer prevention. PUBLIC HEALTH RELEVANCE: This proposed study, based on data and biosamples available from a prospective cohort, will be the first to examine the epigenetic impact of long-term exposure to light at night (night shift work). In light of the recent IARC conclusion that "shift work that involves circadian disruption is probably carcinogenic to humans", along with an increasing body of both epidemiological and biological evidence, the findings from this study may provide possible biological mechanisms for the role of shift work in breast cancer development, and will be valuable in directing future resources to appropriate strategies for primary prevention. Due to the high prevalence of exposure to occupational shift work worldwide, the proposed study has the potential for broad public health implications.